Real time assays for the detection of specific nucleic acids are of great importance, since they enable instant detection of specific RNA/DNA sequences without the need of separating the probe-analyte hybrid from the unbound probe. Molecular beacon probes are a novel tool for real-time nucleic acid analysis (Tyagi & Kramer (1996) Nat. Biotechnol. 14: 303-308; Marras et al., (2006) Clin. Chim. Acta. 363: 48-60; Wang, et al., (2009) Angew. Chem. Int. Ed. Engl. 48: 856-870; Li et al., (2008) Biochem. Biophys. Res. Commun. 373: 457-461; Venkatesan et al., (2008) Chem. Soc. Rev. 37: 648-663).
The traditional molecular beacon is a stem-loop folded oligonucleotide with a fluorophore at the 5′-end and a quencher at its 3′-end. Hybridization to a complementary single-stranded DNA or RNA converts the molecular beacon into the elongated conformation, in which the fluorophore is remote from the quencher, resulting in high fluorescence. The simplicity and elegance of molecular beacon design has made this probe a popular tool for nucleic acid analysis and has served as an inspiration for a number of related assays (see, for example, Du et al., (2003) J. Am. Chem. Soc. 125: 4012-4013; Ye et al., (2004) J. Am. Chem. Soc. 126: 7740-7741; Liu & Lu (2006) Methods Mol. Biol. 335: 275-288; Grossmann et al., (2007) Angew. Chem. Int. Ed. Engl. 46: 5223-5225; Lin et al., (2008) Nucleic Acids Res. 36: e123).
Molecular beacon probes are extensively used in real-time PCR assays (Whitman & Dunbar (2008) Recent Pat. DNA Gene Seq. 2: 20-26) and for RNA monitoring in living cells (Bao et al., (2009) Ann. Rev. Biomed. Eng. 11: 25-47; Tyagi. S. (2009) Nat. Methods 6: 331-338). Importantly, the stem-loop structure not only brings the fluorophore close to the quencher but also improves the probe specificity. Molecular beacon probes discriminate between two nucleic acid sequences that differ by a single nucleotide in a wider temperature range than do linear hybridization probes (Bonnet et al., (1999) Proc. Natl. Acad. Sci. USA. 96: 6171-6176). These properties are of particular importance for single nucleotide polymorphism (SNP) genotyping.
One factor that limits the application of molecular beacon probes in SNP genotyping is their high synthetic cost. Chemical synthesis of a regular molecular beacon probe requires conjugation of an oligonucleotide with two organic dyes, a fluorophore and a quencher. Moreover, at least one round of HPLC purification is required to remove fluorescent impurities, which cause high background fluorescence and reduce both the sensitivity and the dynamic range of the probe. Taking into account that the analysis of each individual SNP requires two molecular beacon probes (each complementary to a specific allele), genotyping thousands of SNPs by molecular beacon probes becomes expensive. Accordingly, a binary approach for nucleic acid recognition (Kolpashchikov D. M. (2005) J. Am. Chem. Soc. 127: 12442-12443; Kolpashchikov D. M. (2006) J. Am. Chem. Soc., 128: 10625-10628; Kolpashchikov D. M. (2007) Chembiochem. 8: 2039-2042; Kolpashchikov D. M. (2008). J. Am. Chem. Soc. 130: 2934-2935; Gerasimova et al., (2010) Chembiochem 11: 811-817) has been developed. In this approach two oligonucleotide probes form short hybrids with the analyte and generate a fluorescent signal upon tertiary complex formation. The approach enables SNP genotyping at room temperature with exceptional specificity.